Phage Titrering: Calculating Dilution Factor & Plate Volume

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In summary, the conversation discusses difficulties with choosing the right values for phage titration. The method involves diluting phages 5X with dilution buffer and adding 1ul of the diluted phage to 200ul of XL1-Blue overnight culture, followed by 2ml of melted top agar and plating the mixture. The dilution factor is 5X and does not include the 200ul of culture and 2ml of agar. The "ul of diluted phage plated" refers to the 1ul of diluted phage added to the other volumes. The conversation also clarifies that pfu refers to plaque forming unit and the volume of phage used for counting should be 1ul.
  • #1
mountain
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hi guys!

i am doing phage titrering but have difficulty in chosen the right values. please give me some ideas. thanks in advance!


here is what we did:
dilute phages 5X with dilution buffer. add 1ul of the diluted phage to 200ul of the XL1-Blue overnight culture. Add 2ml melted top agar. Quickly pour onto a plate. the day after count pfu/ml.

pfu/ml: (number of plaque*dilution factor*10^3ul/ml )/ul of diluted phage plated.

my problem is: what is the dilution factor? is that factor 5 or should i also take 200ul of the XL1-Blue cells and 2ml melted top agar in consideration? (practically it seemed like the phages is diluted more than 5X.)

what is the "ul of diluted phage plated"? is that the 1ul diluted phage i added to the other volumes or is that the total volume (1ul phage+200ul XL1-Blue cells+2ml top agar)?


thanks!
 
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  • #2
5X is your dilution factor and does not include the 200 uL of culture and 2 mL of agar. Also, the 200 uL and 2 mL will not dilute your sample since your plating the whole volume and not just part of it. Therefore your are plating all the phage that are in your 1 uL sample.

pfu means plaque forming unit, so you need to use the volume of phage that you used (1 uL) not the total volume. You looking at counting the number of phage in your stock solution.
 
  • #3
thanks! :approve:
 

1. What is phage titration and why is it important?

Phage titration is a process used to determine the number of active phages (virus that infects bacteria) in a sample. It is important because it allows scientists to measure the concentration of phages in a sample, which can help in the study and understanding of phage biology and their potential use in phage therapy.

2. How do you calculate the dilution factor for phage titration?

The dilution factor for phage titration can be calculated by dividing the volume of the original sample by the volume of the aliquot (sample taken for titration). For example, if you take 1 mL of a 10 mL sample for titration, the dilution factor would be 10 (10 mL/1 mL).

3. What is the purpose of calculating the dilution factor in phage titration?

The dilution factor is important in phage titration as it helps in determining the concentration of phages in the original sample. This information is necessary for accurate calculations of the phage titer (number of phages per unit volume) and for determining the appropriate sample size for plating.

4. How do you calculate the plate volume for phage titration?

The plate volume for phage titration can be calculated by multiplying the number of plates needed by the volume of each plate. For example, if you need 10 plates and each plate has a volume of 0.1 mL, the total plate volume would be 1 mL (10 plates x 0.1 mL).

5. What is the significance of determining the plate volume in phage titration?

The plate volume is important in phage titration as it helps in determining the appropriate number of plates needed to achieve a proper phage lawn (layer of bacteria on the agar surface) for accurate counting of plaques (clear zones where bacteria have been killed by phages). This ensures that the phage titer is measured accurately and can be compared between different samples.

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